36 Extended culture after vitrification-warming helps in spindle recovery of bovine oocytes

Abstract

The meiotic spindle is one of the most vulnerable cytoplasmic organelles when performing oocyte vitrification. It has been proposed that submitting oocytes to a post-warming incubation period in maturation medium helps in the reorganization of microtubules and chromosomes. Our previous experiments found no differences in spindle morphology after submitting vitrified oocytes to a 2-h incubation period. The aim of this experiment was to determine the effect of extended culture on the reorganization of the meiotic spindle of vitrified-warmed bovine oocytes. Oocytes were purchased from a commercial vendor (n = 86) and matured during shipment. In this experiment, three treatments were evaluated: fresh oocytes (F) (n = 30), vitrified-warmed (VW; n = 26), and extended culture (EC; n = 30). Cumulus-oocyte complexes were removed at 18 h of maturation. Fresh oocytes were denuded by vortexing in hyaluronidase (1.5 mg mL⁻¹) and immediately fixed using 4% paraformaldehyde. Oocytes undergoing vitrification were partially denuded by pipetting in hyaluronidase (1.5 mg mL⁻¹). The vitrification protocol consisted of incubation in equilibration solution (7.5% dimethyl sulfoxide + 7.5% ethylene glycol) for 9 min and then in vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose). While in vitrification solution, oocytes were mounted onto a Cryolock and plunged into liquid nitrogen in less than 1 min. Warming was performed by placing a Cryolock into 0.5 M sucrose for 3 min and then into 0.25 M sucrose for 3 min. Finally, oocytes were washed in base medium. The base medium used for cryoprotectant and warming solutions was Dulbecco's phosphate-buffered saline supplemented with 20% fetal bovine serum. Both, vitrification and warming, were performed at 38.5°C. After warming, half of the oocytes were completely denuded and fixed and the other half underwent a 6-h incubation period in maturation medium (IVF-Bioscience). To examine microtubule distribution and chromosome arrangement, fixed oocytes were submitted to an immunostaining protocol using α tubulin antibody (1:100) and anti IgG-Alexa Fluor 488 (1:1000; Thermo Fisher Scientific) and counterstained with Hoechst. The effect of extended culture on the incidence of abnormal microtubule distribution and chromosome arrangement was analysed using logistic regression with a binomial response variable (normal/abnormal). There was no difference in maturation rates among groups (F = 73.3%, VW = 77%, EC = 86.6%; P = 0.43). For microtubule distribution, oocytes fixed immediately after warming had a higher incidence of abnormal spindles (57.7%) when compared with oocytes submitted to extended culture (26.6%; P = 0.02). The most common abnormality seen in oocytes fixed after warming was small and faintly stained spindles. Microtubule distribution in fresh oocytes did not differ from oocytes in the other groups. There were no differences in chromosome arrangement among groups (P = 0.11). Future research will focus on evaluating the benefits that this technique offers to improve development following IVF using vitrified-warmed oocytes.